We have developed a soluble DNA replication system that synthesizes full-sized adenovirus DNA molecules. The extraction of Adenovirus type 2 infected HeLa cell nuclei solubilized 25 percent of the replicating viral DNA and removes greater than 99 percent of the chromosomal DNA from the preparation. Both the extracted viral DNA and the DNA product synthesized in vitro are intact and identical to the adenovirus DNA or its replicating intermediated produced in whole cells. The direction of elongation and the termini of replication in vitro are essentially identical to those observed in intact cells. Similar extracts of cells infected with the DNA negative, temperature sensitive mutant, H5ts125, have been prepared and shown temperature sensitive DNA synthesis in our extract system. The defect in DNA elongation can be corrected by addition of a purified wild type protein whose single polypeptide is 72,000 daltons in size. This 72K peptide is a single-stranded DNA binding protein coded by the viral genome.